Polynucleotides encoding thrombin receptor homologs

ABSTRACT

The present invention provides nucleotide and amino acid sequences that identify and encode a novel thrombin receptor homolog (TRH) expressed in human liver. The present invention also provides for antisense molecules to the nucleotide sequences which encode TRH, diagnostic tests based on TRH encoding nucleic acid molecules, expression vectors for the production of purified TRH, antibodies capable of binding specifically to TRH, hybridization probes or oligonucleotides for the detection of TRH-encoding nucleotide sequences, genetically engineered host cells for the expression of TRH, and antagonists, antibodies and inhibitors with specific binding activity for the polypeptide TRH.

[0001] This application is a continuation application of U.S.application Ser. No. 09/643,383, filed Aug. 21, 2000, entitledPOLYNUCLEOTIDES ENCODING THROMBIN RECEPTOR HOMOLOGS (as amended), whichis a divisional application of U.S. application Ser. No. 09/217,101,filed Dec. 21, 1998 and issued on Nov. 7, 2000 as U.S. Pat. No.6,143,870, entitled THROMBIN RECEPTOR HOMOLOG, which is a divisionalapplication of U.S. application Ser. No. 08/911,320, filed Aug. 14, 1997and issued on Feb. 9, 1999 as U.S. Pat. No. 5,869,633, entitled THROMBINRECEPTOR HOMOLOG POLYNUCLEOTIDE, which is a divisional application ofU.S. application Ser. No. 08/467,125, filed Jun. 6, 1995 and issued onNov. 11, 1997 as U.S. Pat. No. 5,686,597, entitled THROMBIN RECEPTORHOMOLOG, the contents all of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a new human thrombin receptor homolog and the use thereof fro thediagnosis, prevention and treatment of disease.

[0003] The thrombin receptor is a G-protein coupled seven transmembranereceptor (T7G) which is present on platelets, endothelial cells,fibroblasts, mesangial cells, neural cells and smooth muscle cells. Thisreceptor is activated by the irreversible cleavage of the extracellular,amino terminal sequence between arg⁴¹ and ser⁴² by thrombin or anotherserine protease. In normal endothelial cells, activation of the thrombinreceptor stimulates intracellular Gq-protein mediated phosphoinositidemetabolism and Gi-protein mediated adenylate cyclase inhibition.Additionally, endothelial cells secrete endothelin-derived relaxingfactor (EDRF). Subsequently EDRF stimulates soluble guanylate cyclaserelease followed by formation of cyclic GMP and relaxation of smoothmuscle. Another function of EDRF is the inhibition of platelet adhesionand aggregation which is beneficial to blood flow.

[0004] Wilcox et al (1994, Circ Res 75:1029-1038) disclosed that in rataorta smooth muscle, thrombin receptor expression increases soon aftervascular injury and triggers cell proliferation in the neointima.Blocking the thrombin receptor's cleavage site with antibody haltsthrombin-induced cell proliferation. Following cleavage and activation,thrombin receptors become desensitized, and they are internalized forrecycling. Receptor recycling and replacement limit the duration ofthrombin initiated events.

[0005] The thrombin receptor has at least four potential N-glycosylationsites, Asn³⁵, Asn⁶², Asn⁷⁵, and Asn²⁵⁹. The presence of carbohydrategreatly affects the differences in size and predicted mass reported forthrombin receptors. Moreover, glycosylation may account for as much as30% of the mass of these receptors and apparently determines theirdistribution (Brass L F et al (1992) J Biol Chem 267:13795-13798).

[0006] The thrombin receptor is classified with the nonneurokinin T7Greceptors which include many glycoprotein hormone receptors such asthose for luteinizing hormone (LH) and follicle stimulating hormone(FSH). They have very long N-termini, bind a common ligand structuralmotif with low affinity to activate the receptor, and rely on theN-termini and extracellular loops to impart high affinity andspecificity (Bolander F F (1994) Molecular Endocrinology, AcademicPress, San Diego Calif., pp. 162-176).

[0007] They are related to other T7Gs by their seven hydrophobic domainswhich span the plasma membrane and form a bundle of antiparallel αhelices. These transmembrane segments (TMS) are designated by romannumerals I-VII and account for structural and functional features of thereceptor. In most cases, the bundle forms a binding pocket; however,when the binding site must accommodate more bulky molecules, theextracellular N-terminal segment or one or more of the threeextracellular loops participate in binding (Watson S and Arkinstall S(1994) The G-Protein Linked Receptor Facts Book, Academic Press, SanDiego Calif., pages 20-31) and in subsequent induction of conformationalchange in intracellular portions of the receptor. The activated receptorin turn, interacts with an intracellular G-protein complex whichmediates further intracellular signalling activities including; theproduction of second messengers such as cyclic AMP (cAMP), phospholipaseC, inositol triphosphate or ion channel proteins.

[0008] The novel thrombin receptor homolog (trh) which is the subject ofthis patent application was identified among the cDNAs derived from aliver tissue library. Incyte Clone 86700 is most similar to HUMTHRR, thehuman thrombin receptor (Dennington P M and M C Berndt (1994) Clin ExpPharmacol Physiol 21:349-358). Additionally, Incyte 86700 displays aminoacid sequence similarity to platelet activating factor receptor(residues 94-155). These amino acids cover most of TMS III, the secondintracellular loop, and TMS IV. Incyte 86700 was expressed in cells inthe liver which is briefly described below.

[0009] The basic functional unit of the human liver is the lobule, andeach lobule consists of hepatic cellular plates which encircle a centralvein. Small bile canaliculi bisect the plates and empty into ducts foundin septa between the lobules. Hepatic sinusoids associated with thesepta are lined with endothelial and Kupffer cells. Bacteria and organicmatter are removed from the portal blood by the Kupffer cells which alsoexpress high levels of class II major histocompatibility complexproteins and release nitric oxide, interleukin (IL)-1, IL-6, and TNF.

[0010] Although they do not normally produce erythrocytes or divide inadults, liver cells are basically embryonic. If a portion of the liveris destroyed or removed, the remaining parenchyma can regenerate theentire organ. Liver cells are characterized by large amounts ofendoplasmic reticulum which is consistent with their metabolic, storage,endo- and exo-cytotic activities. The liver has three main functions: 1)vascular, for the storage and filtration of blood; 2) metabolic, forregulation of glucose and storage of glycogen and fat; and 3)secretory/excretory, for regulation of hormones and degradation of toxicsubstances.

[0011] The normal blood volume of the liver is 450 ml, and an averageblood flow through the liver is 1450 ml/min. The liver can store anextra 500-1000 ml of blood or supply extra blood after trauma. Half ofthe lymph produced in the body comes from the liver via drainage throughthe spaces of Disse (located below the hepatic cells). High bloodpressure within the liver is a significant factor in edema or ascites.

[0012] Liver cells have a very high rate of metabolism; and theyprocess, synthesize and degrade carbohydrates, fats and proteinssimultaneously. Carbohydrate metabolism consists of storage of glycogen,conversion of fructose and galactose to glucose, and gluconeogenesis.The liver regulates the amount of glucose in the blood in response tohormones.

[0013] Liver cells have special fat-metabolizing functions, theoxidation of fatty acids and the production of lipoproteins,phospholipids and cholesterol. The lipoproteins transport phospholipidsand cholesterol to other areas where they are used to form cellularmembranes and other substances important in cellular function. Most ofthe conversion of carbohydrates and proteins to fat occurs in the liveralthough storage may be in adipose tissue elsewhere in the body. Theliver also stores the fat-soluble vitamins and iron.

[0014] The liver forms 90% of the plasma proteins, particularly albuminand various globulins, at the rate of 15-50 g/day. The liver alsoproduces haptoglobin, ceruloplasmin, and transferrin in response tocytokines. It manufactures nonessential amino acids; and in the presenceof vitamin K, synthesizes accelerator globulin, erythropoietin, and theclotting factors I, II, V, VII, IX and X.

[0015] Lipid-soluble drugs are usually detoxified in the liver. Phase Ireactions involve enzymatic modification of reactive groups on drugmolecules by oxidation, reduction, hydroxylation, sulfoxidation,deamination, dealkylation or methylation. Such modifications deactivatebarbiturates and benzodiazepines and activate cortisone or prednisone.The enzymes responsible for phase I reactions are induced by ethanol andbarbiturates and inhibited by chloramphenicol, cimetidine and ethanol.

[0016] Phase II reactions involve enzymatic conversion of substances totheir acid or salt derivatives, eg, glucuronide, glycine, or sulfate.When the liver is damaged, it is slower in processing of anticonvulsants(phenobarbital), anti-inflammatory agents (acetaminophen orglucocorticoids), tranquilizers (lidocaine, propranolol) or antibiotics(chloramphenicol, tetracycline, or rifampin). In addition, the liverinactivates endogenous hormones by deamination, proteolysis, ordeiodination. Glucocorticoids and aldosterone are reduced to theirtetrahydro derivative and conjugated to glucuronic acid. Testosteroneand estrogen are converted to ketosteroids and conjugated with sulfatesor glucuronic acid.

[0017] Abnormal physiologic or pathologic conditions of the liverinclude fatty liver, which is the excessive accumulation of lipids inhepatocytes in response to injury; jaundice, in which large quantitiesof bilirubin are present in the extracellular fluid; hepatitis, whichare viral infections caused by hepadnaviruses but whose major pathologyresults from host immunologic response; cirrhosis, which is caused byirreversible chronic injury of hepatic parenchyma and progresses tofibrosis; infiltrative diseases such as granulomas and amyloidosis; andadenomas and carcinomas.

[0018] Clinicians currently evaluate bilirubin and urobilinogen levelsto diagnose hemolytic liver disease and assay carcinoembryonic antigenlevel to diagnose metastatic cancer. Depending on its function, an assayfor thrombin receptor homolog could become a diagnostic tool forexcessive fibrosis and resulting liver damage. If the thrombin receptorhomolog is expressed in response to mechanical or chemical injury orinvolved in unnecessary clotting reactions or progressive fibrosis, thenit may also represent an accessible therapeutic target for controllinginflammatory processes in the liver.

[0019] Liver anatomy, physiology, and diseases are reviewed, inter alia,in Guyton, A C (1991) Textbook of Medical Physiology, WB Saunders Co,Philadelphia Pa.; Isselbacher, K J et al (1994) Harrison's Principles ofInternal Medicine, McGraw-Hill, New York N.Y.; and The Merck Manual ofDiagnosis and Therapy (1992) Merck Research Laboratories, Rahway N.J.

SUMMARY OF THE INVENTION

[0020] The subject invention provides a unique nucleotide sequence, SEQID NO: 1, which encodes a novel human thrombin receptor homolog (TRH),SEQ ID NO:2. The cDNA, herein designated trh, was identified and clonedusing Incyte Clone No. 86700 from a liver cDNA library.

[0021] The invention also comprises the use of this TRH or its variantsto intercede in physiologic or pathologic conditions and includediagnosis or therapy of activated or inflamed cells and/or tissues withtrh nucleic acids, fragments or oligomers thereof. Aspects of theinvention include the antisense DNA of trh; cloning or expressionvectors containing trh; host cells or organisms transformed withexpression vectors containing trh; a method for the production andrecovery of purified TRH from host cells; purified protein, TRH, whichcan be used to identify antagonists, antibodies or inhibitors of thereceptor for therapeutic use.

DESCRIPTION OF THE FIGURES

[0022]FIGS. 1A and 1B show the nucleotide (SEQ ID NO:1) and amino acid(SEQ ID NO:2) alignments of the extended sequence for TRH. The oligomersused to extend the nucleotide sequences to full length areXLR=AAGGAGGGCATAATTCCACAATGTG (SEQ ID NO:4) andXLF=AAGGAGGGCATAATTCCACAATGTG (SEQ ID NO:5).

[0023]FIGS. 2A, 2B, and 2C display the alignment of human TRH withHUMTHRR (SEQ ID NO:3), human thrombin receptor. Note the presence of theconserved Arg⁴¹ and Ser⁴² cleavage site.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Definitions

[0025] As used herein, TRH, refers to thrombin receptor homologs,naturally occurring TRHs and active fragments thereof, which haveessentially the amino acid sequence shown in SEQ ID NO:2. In oneembodiment, the polypeptide TRH is encoded by mRNAs transcribed from thecDNA (trh) of SEQ ID NO:1.

[0026] “Active” refers to those forms of TRH which retain the biologicand/or immunologic activities of any naturally occurring TRH.

[0027] “Naturally occurring TRH” refers to TRHs produced by human cellsthat have not been genetically engineered and specifically contemplatesvarious TRHs arising from post-translational modifications of thepolypeptide including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.

[0028] “Derivative” refers to TRHs chemically modified by suchtechniques as ubiquitination, labeling (eg, with radionuclides, variousenzymes, etc.), pegylation (derivatization with polyethylene glycol),and insertion or substitution by chemical synthesis of amino acids suchas ornithine, which do not normally occur in human proteins.

[0029] “Recombinant polypeptide variant” refers to any polypeptidehaving the activity of the TRH polypeptide and differing from naturallyoccurring TRHs by amino acid insertions, deletions, and substitutionscreated using recombinant DNA techniques. Guidance in determining whichamino acid residues may be replaced, added or deleted without abolishingactivities of interest, such as normal signal transduction, may be foundby comparing the sequence of the particular TRH with that of homologouspeptides and minimizing the number of amino acid sequence changes madein highly conserved regions.

[0030] Preferably, amino acid “substitutions” are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, ora threonine with a serine, ie, conservative replacements. “Insertions”or “deletions” are typically in the range of about 1 to 5 amino acids.The variation allowed may be experimentally determined by producing thepeptide synthetically or by systematically making insertions, deletions,or substitutions of nucleotides in a trh molecule using recombinant DNAtechniques and assaying the expressed, recombinant variants foractivity.

[0031] A polypeptide “fragment”, “portion”, or “segment” is a stretch ofamino acid residues of at least 5 amino acids and as many as about 20amino acids, preferably about 9 to 13 amino acids. To be active, any TRHfragment must have sufficient length to display biologic and/orimmunologic activity either alone or as part of a chimeric displaymolecule such as keyhole limpet hemocyanin for antibody production or achimeric test molecule consisting mostly of a purinergic receptor forevaluating activity.

[0032] Where desired, a “signal or leader sequence” can direct thepolypeptide through the membrane of a cell. Such a sequence may benaturally present on the polypeptides of the present invention orprovided from heterologous sources by recombinant DNA techniques.

[0033] “Probe” is an RNA or DNA of sufficient length of use in molecularamplification or hybridization to detect complementary sequences.

[0034] An “oligonucleotide” or “oligomer” is a stretch of nucleotideresidues which has a sufficient number of bases to be used as a primeror probe in a polymerase chain reaction (PCR). Such oligonucleotides areprepared based on the t7g cDNA sequence which is found in the SequenceListing. After appropriate testing to establish reaction conditions andto eliminate false positives, they are used to amplify, reveal orconfirm the presence of an identical or homologous DNA or itstranscribed RNA in a particular cell or tissue. Oligonucleotides oroligomers comprise portions of a DNA sequence having at least about 10nucleotides and as many as about 35 nucleotides, preferably about 25nucleotides.

[0035] “Degenerate oligonucleotide probes” are probes in which minorsubstitutions based on the degeneracy of the genetic code (more than onecodon may specify the same amino acid) have been made. They areparticularly useful in the amplification of a similar natural nucleicacid sequence from chromosomal DNA as described by Walsh P S et al(1992, PCR Methods Appl 1:241-50).

[0036] A “portion” or “fragment” of a polynucleotide or nucleic acidcomprises all or any part of the nucleotide sequence of a t7g havingfewer nucleotides than about 6 kb, preferably fewer than about 1 kbwhich can be labelled and used as probe. A probe may be labelled with aradioactive element, an enzyme, or a chromogenic or fluorogenic marker.After appropriate testing to establish reaction conditions and toeliminate false positives, nucleic acid probes may be used in Southern,northern or in situ hybridizations to determine whether DNA or RNAencoding a particular T7G or T7G homolog are present in a particularcell type, tissue, or organ.

[0037] Although the sequences for nucleic acid probes may be derivedfrom naturally occurring, recombinant or computer consensus sequences,the physical probe may be chemically synthesized, entirely or in part,and labeled by nick translation, Klenow fill-in reaction, PCR or othermethods well known in the art. Probes of the present invention, theirpreparation and/or labeling are elaborated in Sambrook J et al (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor N.Y.; or Ausubel F M et al (1989) Current Protocolsin Molecular Biology, John Wiley & Sons, New York City, bothincorporated herein by reference.

[0038] “Recombinant nucleotide variants” encoding T7Gs may besynthesized or selected by making use of the “redundancy” in the geneticcode. Various codon substitutions, such as the silent changes whichproduce specific restriction sites, may be introduced to optimizecloning into a plasmid or viral vector or to increase expression in aparticular prokaryotic or eukaryotic system. Codon usage-specificmutations may also be introduced or chimeras containing the domains ofrelated peptides added to test or modify the properties of any part ofthe polypeptide, particularly to change ligand-binding affinities,interchain affinities, or degradation/turnover rate.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention provides a unique nucleotide sequenceidentifying a novel homolog of human thrombin receptor which was firstidentified among the cDNAs of a liver library. The sequence for trh isshown in SEQ ID NO:1 and is homologous to but significantly differentfrom the GenBank sequence, HUMTHRR. Because TRH may be expressed incells responding to trauma or infection, the nucleic acid (trh),polypeptide (TRH) and antibodies to TRH are useful in investigations ofand interventions in the normal and abnormal physiologic and pathologicprocesses which regulate cell signalling, immunity, repair, etc.Therefore, an assay for upregulated expression of TRH can acceleratediagnosis and proper treatment of conditions caused by abnormal signaltransduction due to systemic and local infections, traumatic and othertissue damage, hereditary or environmental diseases associated withhypertension, carcinomas, and other pathologic problems.

[0040] The nucleotide sequence encoding TRH (or its complement) hasnumerous other applications in techniques known to those skilled in theart of molecular biology. These techniques include use as hybridizationprobes for Southerns or northerns, use as oligomers for PCR, use forchromosomal and gene mapping, use in the recombinant production of TRH,use in generation of anti-sense DNA or RNA, their chemical analogs andthe like, and use in production of chimeric molecules for selectingagonists, inhibitors or antagonists for design of domain-specifictherapeutic molecules. Uses of the nucleotides encoding TRH disclosedherein are exemplary of known techniques and are not intended to limittheir use in any technique known to a person of ordinary skill in theart. Furthermore, the nucleotide sequences disclosed herein may be usedin molecular biology techniques that have not yet been developed,provided the new techniques rely on properties of nucleotide sequencesthat are currently known, e.g., the triplet genetic code, specific basepair interactions, etc.

[0041] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofTRH-encoding nucleotide sequences, some bearing minimal homology to thenucleotide sequence of any known and naturally occurring gene may beproduced. The invention has specifically contemplated each and everypossible variation of nucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the nucleotide sequence of naturally occurring TRH,and all such variations are to be considered as being specificallydisclosed.

[0042] Although nucleotide sequences which encode TRH and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring TRH gene under stringent conditions, it may beadvantageous to produce nucleotide sequences encoding TRH or itsderivatives possessing a substantially different codon usage. Codons canbe selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding TRH and its derivatives without altering the encoded amino acidsequence include the production of RNA transcripts having more desirableproperties, such as a greater half-life, than transcripts produced fromthe naturally occurring sequence.

[0043] The nucleotide sequence encoding TRH may be joined to a varietyof other nucleotide sequences by means of well established recombinantDNA techniques (Sambrook J et al, supra). Useful nucleotide sequencesfor joining to trh include an assortment of cloning vectors—plasmids,cosmids, lambda phage derivatives, phagemids, and the like—that are wellknown in the art and may be chosen for such characteristics as the sizeinsert they can accommodate, their utility, their fidelity, etc. Othervectors of interest include expression vectors, replication vectors,probe generation vectors, sequencing vectors, YAC and BAC mappingvectors, and the like. In general, these vectors may contain an originof replication functional in at least one organism, convenientrestriction endonuclease sensitive sites, and selectable markers for thehost cell.

[0044] Another aspect of the subject invention is to provide fortrh-specific nucleic acid hybridization probes capable of hybridizingwith naturally occurring nucleotide sequences encoding TRH. Such probesmay also be used for the detection of TRH-encoding sequences and shouldpreferably contain at least 50% of the nucleotides from any particulardomain of interest from this trh encoding sequence. The hybridizationprobes of the subject invention may be derived from the nucleotidesequence of the SEQ ID NO 1 or from genomic sequence including promoter,enhancer elements and introns of the respective naturally occurring trh.Hybridization probes may be labeled by a variety of reporter groups,including radionuclides such as ³²P or ³⁵S, or enzymatic labels such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0045] PCR, as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and4,965,188, provides additional uses for oligonucleotides based upon thenucleotide sequences which encode TRH. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or may be a mixtureof both and comprise a discrete nucleotide sequence for diagnostic useor a degenerate pool of possible sequences for identification of closelyrelated T7G sequences.

[0046] Full length genes may be cloned from known sequence using a newmethod which employs XL-PCR (Perkin-Elmer, Foster City, Calif.) toamplify long pieces of DNA. This method was developed to allow a singleresearcher to process multiple genes (up to 20 or more) at a time and toobtain an extended (possibly full-length) sequence within 6-10 days. Itreplaces current methods which use labelled probes to screen librariesand allow one researcher to process only about 3-5 genes in 14-40 days.

[0047] In the first step, which can be performed in about two days,primers are designed and synthesized based on a known partial sequence.In step 2, which takes about six to eight hours, the sequence isextended by PCR amplification of a selected library. Steps 3 and 4,which take about one day, are purification of the amplified cDNA and itsligation into an appropriate vector. Step 5, which takes about one day,involves transforming and growing host bacteria. In step 6, which takesapproximately five hours, PCR is used to screen bacterial clones forextended sequence. The final steps, which take about one day, involvethe preparation and sequencing of selected clones. If the full lengthcDNA has not been obtained, the entire procedure is repeated usingeither the original library or some other preferred library. Thepreferred library may be one that has been size-selected to include onlylarger cDNAs or may consist of single or combined commercially availablelibraries, eg. lung, liver, heart and brain from Gibco/BRL (GaithersburgMd.). The cDNA library may have been prepared with oligo dT or randomprimers. The advantage of using random primed libraries is that theywill have more sequences which contain 5′ ends of genes. A randomlyprimed library may be particularly useful if an oligo dT library doesnot yield a complete gene. Obviously, the larger the protein, the lesslikely it is that the complete gene will be found in a single plasmid.

[0048] Other means for producing hybridization probes for T7G DNAsinclude the cloning of nucleic acid sequences encoding TRH or itsderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art and are commercially available and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate labeled nucleotides.

[0049] It is now possible to produce a DNA sequence, or portionsthereof, encoding TRH and/or its derivatives entirely by syntheticchemistry. Such molecules can be inserted into any of the many availablevectors using reagents and methods that are known in the art at the timeof the filing of this application. Moreover, synthetic chemistry may beused to introduce mutations into the trh sequences or any portionthereof.

[0050] The nucleotide sequence can be used to develop an assay to detectactivation, inflammation, or disease associated with abnormal levels ofTRH expression. The nucleotide sequence can be labeled by methods knownin the art and added to a fluid or tissue sample from a patient. Afteran incubation period sufficient to effect hybridization, the sample iswashed with a compatible fluid which contains a visible marker, a dye orother appropriate molecule(s), if the nucleotide has been labeled withan enzyme. After the compatible fluid is rinsed off, the dye isquantitated and compared with a standard. If the amount of dye issignificantly elevated (or lowered, as the case may be), the nucleotidesequence has hybridized with the sample, and the assay indicates anabnormal condition such as inflammation or disease.

[0051] The nucleotide sequence for trh can be used to constructhybridization probes for mapping the gene. The nucleotide sequenceprovided herein may be mapped to a chromosome and specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York City.

[0052] Fluorescent in situ hybridization of chromosomal preparations andother physical chromosome mapping techniques may be correlated withadditional genetic map data. Examples of genetic map data can be foundin the 1994 Genome Issue of Science (265:1981f). Correlation between thelocation of trh on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) can help delimit the region of DNAassociated with that genetic disease. The nucleotide sequence of thesubject invention may be used to detect differences in gene sequencebetween normal and carrier or affected individuals.

[0053] The nucleotide sequence encoding TRH may be used to producepurified TRH using well known methods of recombinant DNA technology.Among the many publications that teach methods for the expression ofgenes after they have been isolated is Goeddel (1990) Gene ExpressionTechnology, Methods and Enzymology, Vol 185, Academic Press, SanùDiegoCalif. TRH may be expressed in a variety of host cells, eitherprokaryotic or eukaryotic. Host cells may be from the same species inwhich trh nucleotide sequences are endogenous or from a differentspecies. Advantages of producing TRH by recombinant DNA technologyinclude obtaining adequate amounts of the protein for purification andthe availability of simplified purification procedures.

[0054] Cells transformed with DNA encoding TRH may be cultured underconditions suitable for the expression of TRH and recovery of theprotein from the cell culture. TRH produced by a recombinant cell may besecreted or may be contained intracellularly depending on the particulargenetic construction used. In general, it is more convenient to preparerecombinant proteins in secreted form. Purification steps vary with theproduction process and the particular protein produced.

[0055] Various methods for the isolation of TRH polypeptide may beaccomplished by procedures well known in the art. For example, such apolypeptide may be purified by immunoaffinity chromatography byemploying the antibodies provided by the present invention. Variousother methods of protein purification well known in the art includethose described in Deutscher M (1990) Methods in Enzymology, Vol 182,Academic Press, San Diego Calif.; and in Scopes R (1982) ProteinPurification: Principles and Practice, Springer-Verlag, New York City,both incorporated herein by reference.

[0056] In addition to recombinant production, fragments of TRH may beproduced by direct peptide synthesis using solid-phase techniques(Stewart et al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, SanFrancisco Calif.; Merrifield J (1963) J Am Chem Soc 85:2149-2154). Invitro protein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (ABI, Foster City, Calif.)in accordance with the instructions provided by the manufacturer.Various fragments of TRH may be chemically synthesized separately andcombined using chemical methods to produce the full length polypeptide.

[0057] TRH for antibody induction does not require biological activity;however, the protein must be immunogenic. Peptides used to inducespecific antibodies may have an amino acid sequence consisting of atleast five amino acids, preferably at least 10 amino acids. They shouldmimic an exposed structural portion or epitope of the amino acidsequence of the polypeptide and may contain the entire amino acidsequence of a small domain of TRH. Short stretches of TRH amino acidsmay be fused with those of another protein such as keyhole limpethemocyanin, and antibody produced against the fusion protein.

[0058] Antibodies specific for TRH may be produced by inoculation of anappropriate animal with the polypeptide or an antigenic fragment. Anantibody is specific for TRH if it is specific for an immunogenicepitope of the polypeptide and binds to at least part of the natural orrecombinant protein. Antibody production includes not only thestimulation of an immune response by injection into animals, but alsoanalogous steps in the production of synthetic antibodies or otherspecific-binding molecules such as the screening of recombinantimmunoglobulin libraries (Orlandi R et al (1989) PNAS 86:3833-3837; HuseW D et al (1989) Science 256:1275-1281) or the in vitro stimulation oflymphocyte populations. Current technology (Winter G and Milstein C(1991) Nature 349:293-299) provides for a number of highly specificbinding reagents based on the principles of antibody formation. Thesetechniques may be adapted to produce molecules specifically bindingparticular domains of TRH.

[0059] An additional embodiment of the subject invention is the use ofTRH specific antibodies or the like as bioactive agents to treatabnormal signal transduction due to systemic and local infections,traumatic and other tissue damage, hereditary or environmental diseasesassociated with hypertension, carcinomas, and other pathologic problems.

[0060] Bioactive compositions comprising agonists, antagonists, orinhibitors of TRH may be administered in a suitable therapeutic dosedetermined by any of several methodologies including clinical studies onmammalian species to determine maximum tolerable dose and on normalhuman subjects to determine safe dosage. Additionally, the bioactiveagent may be complexed with a variety of well established compounds orcompositions which enhance stability or pharmacological properties suchas half-life. It is contemplated that a therapeutic, bioactivecomposition may be delivered by intravenous infusion into thebloodstream or any other effective means which could be used fortreatment.

[0061] The examples below are provided to describe the subjectinvention. These examples are provided by way of illustration and arenot included for the purpose of limiting the invention.

EXAMPLES

[0062] I Isolation of mRNA and Construction of the cDNA Library

[0063] The TRH sequence of this application was first identified inIncyte clone No. 86700 among the sequences comprising the human liverlibrary. The human cells used for this library came from a 49 year oldhuman male (Catalogue # 937224; Stratagene, LaJolla Calif.). cDNAsynthesis for the cDNA library was primed with oligo dT, and syntheticadaptor oligonucleotides were ligated onto cDNA ends enabling itsinsertion into UNIZAP vector system (Stratagene). This allowed highefficiency unidirectional (sense orientation) lambda libraryconstruction and the convenience of a plasmid system with blue/whitecolor selection to detect clones with cDNA insertions.

[0064] The cDNA library can be screened with either DNA probes orantibody probes and the PBLUESCRIPT phagemid (Stratagene) can be rapidlyexcised in vivo. The phagemid allows the use of a plasmid system foreasy insert characterization, sequencing, site-directed mutagenesis, thecreation of unidirectional deletions and expression of fusionpolypeptides. The custom-constructed library phage particles wereinfected into XL1-BLUE E. coli host strain (Stratagene), which has ahigh transformation efficiency, increasing the probability of obtainingrare, under-represented clones in the cDNA library. Alternativeunidirectional vectors include but are not limited to pcDNAI(Invitrogen, San Diego Calif.) and pSHlox-1 (Novagen, Madison Wis.).

[0065] II Isolation of cDNA Clones

[0066] The phagemid forms of individual cDNA clones were obtained by thein vivo excision process, in which the host bacterial strain wascoinfected with both the library phage and an fl helper phage.Polypeptides or enzymes derived from both the library-containing phageand the helper phage nicked the DNA, initiated new DNA synthesis fromdefined sequences on the target DNA and created a smaller, singlestranded circular phagemid DNA molecule that included all DNA sequencesof the PBLUESCRIPT plasmid and the cDNA insert. The phagemid DNA wassecreted from the cells and purified, then used to re-infect fresh hostcells, where double stranded phagemid DNA was produced. Because thephagemid carries the gene for β-lactamase, the newly transformedbacteria were selected on medium containing ampicillin.

[0067] Phagemid DNA was purified using the MAGIC MINIPREPS DNAPurification System (Catalogue #A7100; Promega Corp, Madison, Wis.).This small-scale process provides a simple and reliable method forlysing the bacterial cells and rapidly isolating purified phagemid DNAusing a proprietary DNA-binding resin. Phagemid DNA was also purifiedusing the QIAWELL-8 plasmid purification system (QIAGEN Inc, ChatsworthCalif.). This product line provides a convenient, rapid high-throughputmethod for lysing the bacterial cells and isolating highly purifiedphagemid DNA using QIAGEN anion-exchange resin particles. Followingeither purification method, the DNA was eluted and prepared forsequencing and other analytical manipulations.

[0068] III Sequencing of cDNA Clones

[0069] The cDNA inserts from random isolates of the liver library weresequenced in part. Methods for DNA sequencing are well known in the art.Conventional enzymatic methods employed DNA polymerase Klenow fragment,SEQUENASE (US Biochemical Corp, Cleveland Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest. Methods have been developed for the use of bothsingle- and double-stranded templates. The chain termination reactionproducts were electrophoresed on urea-acrylamide gels and detectedeither by autoradiography (for radionuclide-labeled precursors) or byfluorescence (for fluorescent-labeled precursors). Recent improvementsin mechanized reaction preparation, sequencing and analysis using thefluorescent detection method have permitted expansion in the number ofsequences that can be determined per day using machines such as theCatalyst 800 and the Applied Biosystems 377 or 373 DNA sequencers.

[0070] IV Homology Searching of cDNA Clones and Deduced Proteins

[0071] Each sequence so obtained was compared to sequences in GenBankusing a search algorithm developed by Applied Biosystems andincorporated into the INHERIT 670 sequence analysis. In this algorithm,Pattern Specification Language (developed by TRW Inc., Los AngelesCalif.) was used to determine regions of homology. The three parametersthat determine how the sequence comparisons run were window size, windowoffset, and error tolerance. Using a combination of these threeparameters, the DNA database was searched for sequences containingregions of homology to the query sequence, and the appropriate sequenceswere scored with an initial value. Subsequently, these homologousregions were examined using dot matrix homology plots to distinguishregions of homology from chance matches. Smith-Waterman alignments wereused to display the results of the homology search.

[0072] Peptide and protein sequence homologies were ascertained usingthe INHERIT 670 sequence analysis in a way similar to that used in DNAsequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

[0073] Alternatively, BLAST, which stands for Basic Local AlignmentSearch Tool, is used to search for local sequence alignments (Altschul SF (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol215:403-10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. Whereas it is ideal for matcheswhich do not contain gaps, it is inappropriate for performingmotif-style searching. The fundamental unit of BLAST algorithm output isthe High-scoring Segment Pair (HSP).

[0074] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0075] V Identification, Full Length Cloning, Sequencing and Translation

[0076] Analysis of INHERIT results from randomly picked and sequencedportions of clones from liver library identified Incyte 86700 as ahomolog of the thrombin receptor, HUMTHRR. The cDNA insert comprisingIncyte 86700 was fully sequenced and used as the basis for cloning thefull length cDNA.

[0077] The cDNA of Incyte 86700 was extended to full length using amodified XL-PCR (Perkin Elmer) procedure. Primers were designed based onknown sequence; one primer was synthesized to initiate extension in theantisense direction (XLR=TGCCTTCCGTTG CTGTATAGACCG) and the other toextend sequence in the sense direction (XLF=AAGGAGGGCATAATTCCACAATGTG).The primers allowed the sequence to be extended “outward” generatingamplicons containing new, unknown nucleotide sequence for the gene ofinterest. The primers were designed using OLIGO 4.0 (NationalBiosciences Inc, Plymouth Minn.) to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations wereavoided.

[0078] The liver cDNA library was used as a template, and XLR and XLFprimers were used to extend and amplify the 86700 sequence. By followingthe instructions for the XL-PCR kit and thoroughly mixing the enzyme andreaction mix, high fidelity amplification is obtained. Beginning with 25pMol of each primer and the recommended concentrations of all othercomponents of the kit, PCR was performed using the MJ thermocyclerPTC200 (MJ Research, Watertown Mass.) and the following parameters: Step1 94° C. for 60 sec (initial denaturation) Step 2 94° C. for 15 sec Step3 65° C. for 1 min Step 4 68° C. for 7 min Step 5 Repeat step 2-4 for 15additional times Step 6 94° C. for 15 sec Step 7 65° C. for 1 min Step 868° C. for 7 min + 15 sec/cycle Step 9 Repeat step 6-8 for 11 additionaltimes Step 10 72° C. for 8 min Step 11 4° C. (and holding)

[0079] At the end of 28 cycles, 50 μl of the reaction mix was removed;and the remaining reaction mix was run for an additional 10 cycles asoutlined below: Step 1 94° C. for 15 sec Step 2 65° C. for 1 min Step 368° C. for (10 min + 15 sec)/cycle Step 4 Repeat step 1-3 for 9additional times Step 5 72° C. for 10 min

[0080] A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Although all extensions potentally contain a full length gene, some ofthe largest products or bands were selected and cut out of the gel.Further purification involved using a commercial gel extraction methodsuch as QIAQUICK (QIAGEN Inc, Chatsworth Calif.). After recovery of theDNA, Klenow enzyme was used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitated religation and cloning.

[0081] After ethanol precipitation, the products were redissolved in 13μl of ligation buffer. Then, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture was plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing carbenicillin at 25 mg/L. The following day, 12 colonies wererandomly picked from each plate and cultured in 150 μl of liquidLB/carbenicillin medium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture was transferred into a nonsterile96-well plate and after dilution 1:10 with water, 5 μl of each samplewas transferred into a PCR array.

[0082] For PCR amplification, 15 μl of concentrated PCR reaction mix(1.33×) containing 0.75 units of Taq polymerase, a vector primer and oneor both of the gene specific primers used for the extension reactionwere added to each well. Amplification was performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 times Step 6 72° C. for 180 sec Step 7 4° C. (and holding)

[0083] Aliquots of the PCR reactions were run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products werecompared to the original partial cDNAs, and appropriate clones wereselected, ligated into plasmid and sequenced.

[0084] The cDNA (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequences forhuman TRH are shown in FIGS. 1A and 1B. When the three possibletranslations of TRH were searched against protein databases such asSwissProt and PIR, no exact matches were found. FIGS. 2A, 2B, and 2Cshow the comparison between the trh and HUMTHRR sequences.

[0085] VI Antisense analysis

[0086] Knowledge of the correct, complete cDNA sequence of TRH enablesits use as a tool for antisense technology in the investigation of genefunction. Oligonucleotides, cDNA or genomic fragments comprising theantisense strand of trh can be used either in vitro or in vivo toinhibit expression of the mRNA. Such technology is now well known in theart, and antisense molecules can be designed at various locations alongthe nucleotide sequences. By treatment of cells or whole test animalswith such antisense sequences, the gene of interest can be effectivelyturned off. Frequently, the function of the gene can be ascertained byobserving behavior at the intracellular, cellular, tissue or organismallevel (eg, lethality, loss of differentiated function, changes inmorphology, etc.).

[0087] In addition to using sequences constructed to interrupttranscription of a particular open reading frame, modifications of geneexpression can be obtained by designing antisense sequences to intronregions, promoter/enhancer elements, or even to trans-acting regulatorygenes. Similarly, inhibition can be achieved using Hogeboom base-pairingmethodology, also known as “triple helix” base pairing.

[0088] VII Expression of TRH

[0089] Expression of trh may be accomplished by subcloning the cDNAsinto appropriate expression vectors and transfecting the vectors intoanalogous expression hosts. In this particular case, the cloning vectorpreviously used for the generation of the cDNA library also provides fordirect expression of trh sequences in E. coli. Upstream of the cloningsite, this vector contains a promoter for β-galactosidase, followed bysequence containing the amino-terminal Met and the subsequent 7 residuesof β-galactosidase. Immediately following these eight residues is anengineered bacteriophage promoter useful for artificial priming andtranscription and a number of unique restriction sites, including EcoRI, for cloning.

[0090] Induction of the isolated, transfected bacterial strain with IPTGusing standard methods will produce a fusion protein corresponding tothe first seven residues of β-galactosidase, about 15 residues of“linker”, and the peptide encoded within the cDNA. Since cDNA cloneinserts are generated by an essentially random process, there is onechance in three that the included cDNA will lie in the correct frame forproper translation. If the cDNA is not in the proper reading frame, itcan be obtained by deletion or insertion of the appropriate number ofbases by well known methods including in vitro mutagenesis, digestionwith exonuclease III or mung bean nuclease, or the inclusion of anoligonucleotide linker of appropriate length.

[0091] The trh cDNA can be shuttled into other vectors known to beuseful for expression of protein in specific hosts. Oligonucleotideprimers containing cloning sites as well as a segment of DNA (about 25bases) sufficient to hybridize to stretches at both ends of the targetcDNA can be synthesized chemically by standard methods. These primerscan then be used to amplify the desired gene segment by PCR. Theresulting gene segment can be digested with appropriate restrictionenzymes under standard conditions and isolated by gel electrophoresis.Alternately, similar gene segments can be produced by digestion of thecDNA with appropriate restriction enzymes. Using appropriate primers,segments of coding sequence from more than one gene can be ligatedtogether and cloned in appropriate vectors. It is possible to optimizeexpression by construction of such chimeric sequences.

[0092] Suitable expression hosts for such chimeric molecules include,but are not limited to, mammalian cells such as Chinese Hamster Ovary(CHO) and human 293 cells, insect cells such as Sf9 cells, yeast cellssuch as Saccharomyces cerevisiae, and bacteria such as E. coli. For eachof these cell systems, a useful expression vector may also include anorigin of replication to allow propagation in bacteria and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowplasmid selection in bacteria. In addition, the vector may include asecond selectable marker such as the neomycin phosphotransferase gene toallow selection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts may require RNA processing elements such as3′ polyadenylation sequences if such are not part of the cDNA ofinterest.

[0093] Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and includeMMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tacand T7 promoters for bacterial hosts; and alpha factor, alcohol oxidaseand PGH promoters for yeast. Transcription enhancers, such as the roussarcoma virus enhancer, may be used in mammalian host cells. Oncehomogeneous cultures of recombinant cells are obtained through standardculture methods, large quantities of recombinantly produced TRH can berecovered from the conditioned medium and analyzed using chromatographicmethods known in the art.

[0094] VIII Isolation of Recombinant TRH

[0095] TRH may be expressed as a chimeric protein with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and the trh sequence may be useful to facilitate expression ofTRH.

[0096] IX Testing of Chimeric T7Gs

[0097] Functional chimeric T7Gs may be constructed by combining theextracellular receptive sequences of a new isoform with thetransmembrane and intracellular segments of a known isoform for testingpurposes. This concept was demonstrated by Kobilka et al (1988, Science240:1310-1316) who created a series of chimeric α2-β32 adrenergicreceptors (AR) by inserting progressively greater amounts of a2-ARtransmembrane sequence into β2-AR. The binding activity of knownagonists changed as the molecule shifted from having more a2 than β2conformation, and intermediate constructs demonstrated mixedspecificity. The specificity for binding antagonists, however,correlated with the source of the domain VII. The importance of T7Gdomain VII for ligand recognition was also found in chimeras utilizingtwo yeast a-factor receptors and is significant because the yeastreceptors are classified as miscellaneous receptors. Thus, thefunctional role of specific domains appears to be preserved throughoutthe T7G family regardless of category.

[0098] In parallel fashion, internal segments or cytoplasmic domainsfrom a particular isoform may be exchanged with the analogous domains ofa known T7G and used to identify the structural determinants responsiblefor coupling the receptors to trimeric G-proteins (Dohlman et al (1991)Annu Rev Biochem 60:653-88). A chimeric receptor in which domains V, VI,and the intracellular connecting loop from β2-AR were substituted intoa2-AR was shown to bind ligands with a2-AR specificity, but to stimulateadenylate cyclase in the manner of β2-AR. This demonstrates that foradrenergic-type receptors, G-protein recognition is present in domains Vand VI and their connecting loop. The opposite situation was predictedand observed for a chimera in which the V->VI loop from a1-AR replacedthe corresponding domain on β2-AR and the resulting receptor boundligands with β2-AR specificity and activated G-protein-mediatedphosphatidylinositol turnover in the a1-AR manner. Finally, chimerasconstructed from muscarinic receptors also demonstrated that V→VI loopis the major determinant for specificity of G-protein activity (BolanderFF, supra).

[0099] Chimeric or modified T7Gs containing substitutions in theextracellular and transmembrane regions have shown that these portionsof the receptor determine ligand binding specificity. For example, twoSer residues conserved in domain V of all adrenergic and D catecholamineT7G receptors are necessary for potent agonist activity. These serinesare believed to form hydrogen bonds with the catechol moiety of theagonists within the T7G binding site. Similarly, an Asp residue presentin domain III of all T7Gs which bind biogenic amines is believed to forman ion pair with the ligand amine group in the T7G binding site.

[0100] Functional, cloned T7Gs may be expressed in heterologousexpression systems and their biological activity assessed (Marullo et al(1988) Proc Natl Acad Sci USA 85:7551-55; King et al (1990) Science250:121-123). One heterologous system introduces genes for a mammalianT7G and a mammalian G-protein into yeast cells. The T7G was shown tohave appropriate ligand specificity and affinity and trigger appropriatebiological activation—growth arrest and morphological changes—of theyeast cells. THR domains may be tested in a similar manner.

[0101] X Production of TRH Specific Antibodies

[0102] Two approaches are utilized to raise antibodies to TRH, and eachapproach is useful for generating either polyclonal or monoclonalantibodies. In one approach, denatured protein from reverse phase HPLCseparation is obtained in quantities up to 75 mg. This denatured proteincan be used to immunize mice or rabbits using standard protocols; about100 micrograms are adequate for immunization of a mouse, while up to 1mg might be used to immunize a rabbit. For identifying mouse hybridomas,the denatured protein can be radioiodinated and used to screen potentialmurine B-cell hybridomas for those which produce antibody. Thisprocedure requires only small quantities of protein, such that 20 mgwould be sufficient for labeling and screening of several thousandclones.

[0103] In the second approach, the amino acid sequence of an appropriateTRH domain, as deduced from translation of the cDNA, is analyzed todetermine regions of high immuno-genicity. Oligopeptides comprisingappropriate hydrophilic regions, as illustrated in FIG. 3, aresynthesized and used in suitable immunization protocols to raiseantibodies. Analysis to select appropriate epitopes is described byAusubel F M et al (supra). The optimal amino acid sequences forimmunization are usually at the C-terminus, the N-terminus and thoseintervening, hydrophilic regions of the polypeptide which are likely tobe exposed to the external environment when the protein is in itsnatural conformation.

[0104] Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, StLouis Mo.) by reaction with M-maleimidoben-zoyl-N-hydroxysuccinimideester (MBS; Ausubel F M et al, supra). If necessary, a cysteine may beintroduced at the N-terminus of the peptide to permit coupling to KLH.Rabbits are immunized with the peptide-KLH complex in complete Freund'sadjuvant. The resulting antisera are tested for antipeptide activity bybinding the peptide to plastic, blocking with 1% bovine serum albumin,reacting with antisera, washing and reacting with labeled (radioactiveor fluorescent), affinity purified, specific goat anti-rabbit IgG.

[0105] Hybridomas may also be prepared and screened using standardtechniques. Hybridomas of interest are detected by screening withlabeled TRH to identify those fusions producing the monoclonal antibodywith the desired specificity. In a typical protocol, wells of plates(FAST; Becton-Dickinson, Palo Alto Calif.) are coated during incubationwith affinity purified, specific rabbit anti-mouse (or suitableanti-species Ig) antibodies at 10 mg/ml. The coated wells are blockedwith 1% BSA, washed and incubated with supernatants from hybridomas.After washing the wells are incubated with labeled TRH at 1 mg/ml.Supernatants with specific antibodies bind more labeled TRH than isdetectable in the background. Then clones producing specific antibodiesare expanded and subjected to two cycles of cloning at limitingdilution. Cloned hybridomas are injected into pristane-treated mice toproduce ascites, and monoclonal antibody is purified from mouse asciticfluid by affinity chromatography on Protein A. Monoclonal antibodieswith affinities of at least 10⁸ M⁻¹, preferably 10⁹ to 10¹⁰ or stronger,will typically be made by standard procedures as described in Harlow andLane (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.; and in Goding (1986) MonoclonalAntibodies: Principles and Practice, Academic Press, New York City, bothincorporated herein by reference.

[0106] XI Diagnostic Test Using TRH Specific Antibodies

[0107] Particular TRH antibodies are useful for investigating signaltransduction and the diagnosis of infectious or hereditary conditionswhich are characterized by differences in the amount or distribution ofTRH or downstream products of an active signalling cascade. Since TRHwas found in a human liver library, it appears to be upregulated in celltypes mainly involved in immune protection or defense.

[0108] Diagnostic tests for TRH include methods utilizing antibody and alabel to detect TRH in human body fluids, membranes, cells, tissues orextracts of such. The polypeptides and antibodies of the presentinvention may be used with or without modification. Frequently, thepolypeptides and antibodies will be labeled by joining them, eithercovalently or noncovalently, with a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and have been reported extensively in both the scientific andpatent literature. Suitable labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent agents, chemiluminescentagents, magnetic particles and the like. Patents teaching the use ofsuch labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinantimmunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567,incorporated herein by reference.

[0109] A variety of protocols for measuring soluble or membrane-boundTRH, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson TRH is preferred, but a competitive binding assay may be employed.These assays are described, among other places, in Maddox, D E et al(1983, J Exp Med 158:1211f).

[0110] XII Purification of Native TRH Using Specific Antibodies

[0111] Native or recombinant TRH can be purified by immunoaffinitychromatography using antibodies specific for TRH. In general, animmunoaffinity column is constructed by covalently coupling the anti-TRHantibody to an activated chromatographic resin.

[0112] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated Sepharose (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0113] Such immunoaffinity columns may be utilized in the purificationof TRH by preparing a fraction from cells containing TRH in a solubleform. This preparation may be derived by solubilization of whole cellsor of a subcellular fraction obtained via differential centrifugation(with or without addition of detergent) or by other methods well knownin the art. Alternatively, soluble TRH containing a signal sequence maybe secreted in useful quantity into the medium in which the cells aregrown.

[0114] A soluble TRH-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of TRH (eg, high ionic strengthbuffers in the presence of detergent). Then, the column is eluted underconditions that disrupt antibody/TRH binding (eg, a buffer of pH 2-3 ora high concentration of a chaotrope such as urea or thiocyanate ion),and TRH is collected.

[0115] XIII Drug Screening

[0116] This invention is particularly useful for screening therapeuticcompounds by using TRH or binding fragments thereof in any of a varietyof drug screening techniques. The polypeptide or fragment employed insuch a test may either be free in solution, affixed to a solid support,borne on a cell surface or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the polypeptide orfragment. Drugs are screened against such transformed cells incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,the formation of complexes between TRH and the agent being tested.Alternatively, one can examine the diminution in complex formationbetween TRH and a receptor caused by the agent being tested.

[0117] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect signal transduction. Thesemethods, well known in the art, comprise contacting such an agent withTRH polypeptide or a fragment thereof and assaying (i) for the presenceof a complex between the agent and the TRH polypeptide or fragment, or(ii) for the presence of a complex between the TRH polypeptide orfragment and the cell. In such competitive binding assays, the TRHpolypeptide or fragment is typically labeled. After suitable incubation,free TRH polypeptide or fragment is separated from that present in boundform, and the amount of free or uncomplexed label is a measure of theability of the particular agent to bind to TRH or to interfere with theTRH and agent complex.

[0118] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to the TRHpolypeptides and is described in detail in European Patent Application84/03564, published on Sep. 13, 1984, incorporated herein by reference.Briefly stated, large numbers of different small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with TRH polypeptide andwashed. Bound TRH polypeptide is then detected by methods well known inthe art. Purified TRH can also be coated directly onto plates for use inthe aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

[0119] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding TRHspecifically compete with a test compound for binding to TRHpolypeptides or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with TRH.

[0120] XIV Rational Drug Design

[0121] The goal of rational drug design is to produce structural analogsof biologically active polypeptides of interest or of small moleculeswith which they interact, eg, agonists, antagonists, or inhibitors. Anyof these examples can be used to fashion drugs which are more active orstable forms of the polypeptide or which enhance or interfere with thefunction of a polypeptide in vivo (Hodgson J (1991) Bio/Technology9:19-21, incorporated herein by reference).

[0122] In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design efficient inhibitors. Usefulexamples of rational drug design may include molecules which haveimproved activity or stability as shown by Braxton S and Wells J A(1992, Biochemistry 31:7796-7801) or which act as inhibitors, agonists,or antagonists of native peptides as shown by Athauda S B et al (1993 JBiochem 113:742-46), incorporated herein by reference.

[0123] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids is expected to be an analog of the original receptor. Theanti-id can then be used to identify and isolate peptides from banks ofchemically or biologically produced peptides. The isolated peptides thenact as the pharmacore.

[0124] By virtue of the present invention, sufficient amount ofpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the TRH amino acidsequence provided herein will provide guidance to those employingcomputer modeling techniques in place of or in addition to x-raycrystallography.

[0125] XV Identification of Other Members of the Signal TransductionComplex

[0126] The inventive purified TRH is a research tool for identification,characterization and purification of interacting G or other signaltransduction pathway proteins. Radioactive labels are incorporated intoa selected TRH domain by various methods known in the art and used invitro to capture interacting molecules. A preferred method involveslabeling the primary amino groups in TRH with ¹²⁵I Bolton-Hunter reagent(Bolton, A E and Hunter, W M (1973) Biochem J 133: 529). This reagenthas been used to label various molecules without concomitant loss ofbiological activity (Hebert C A et al (1991) J Biol Chem 266: 18989;McColl S et al (1993) J Immunol 150:4550-4555).

[0127] Labeled TRH is useful as a reagent for the purification ofmolecules with which it interacts. In one embodiment of affinitypurification, membrane-bound TRH is covalently coupled to achromatography column. Cell-free extract derived from liver cells orputative target cells is passed over the column, and molecules withappropriate affinity bind to TRH. The TRH-complex is recovered from thecolumn, dissociated and the recovered molecule is subjected toN-terminal protein sequencing. This amino acid sequence is then used toidentify the captured molecule or to design degenerate oligonucleotideprobes for cloning the relevant gene from an appropriate cDNA library.

[0128] In an alternate method, antibodies are raised against TRH,specifically monoclonal antibodies. The monoclonal antibodies arescreened to identify those which inhibit the binding of labeled TRH.These monoclonal antibodies are then used therapeutically.

[0129] XVI Use and Administration of Antibodies, Inhibitors, orAntagonists

[0130] Antibodies, inhibitors, or antagonists of TRH (or othertreatments to limit signal transduction, LST), can provide differenteffects when administered therapeutically. LSTs will be formulated in anontoxic, inert, pharmaceutically acceptable aqueous carrier mediumpreferably at a pH of about 5 to 8, more preferably 6 to 8, although thepH may vary according to the characteristics of the antibody, inhibitor,or antagonist being formulated and the condition to be treated.Characteristics of LSTs include solubility of the molecule, half-lifeand antigenicity/immunogenicity; these and other characteristics may aidin defining an effective carrier. Native human proteins are preferred asLSTs, but organic or synthetic molecules resulting from drug screens maybe equally effective in particular situations.

[0131] LSTs may be delivered by known routes of administration includingbut not limited to topical creams and gels; transmucosal spray andaerosol; transdermal patch and bandage; injectable, intravenous andlavage formulations; and orally administered liquids and pillsparticularly formulated to resist stomach acid and enzymes. Theparticular formulation, exact dosage, and route of administration willbe determined by the attending physician and will vary according to eachspecific situation.

[0132] Such determinations are made by considering multiple variablessuch as the condition to be treated, the LST to be administered, and thepharmacokinetic profile of the particular LST. Additional factors whichmay be taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time and frequency ofadministration, drug combination, reaction sensitivities, andtolerance/response to therapy. Long acting LST formulations might beadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular LST.

[0133] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature. See U.S. Pat. Nos. 4,657,760;5,206,344; or 5,225,212. Those skilled in the art will employ differentformulations for different LSTs. Administration to cells such as nervecells necessitates delivery in a manner different from that to othercells such as vascular endothelial cells.

[0134] It is contemplated that abnormal signal transduction, trauma, ordiseases which trigger TRH activity may be treatable with LSTs. Theseconditions or diseases may be specifically diagnosed by the testsdiscussed above, and such testing should be performed in suspected casesof systemic and local infections, traumatic and other tissue damage,hereditary or environmental diseases associated with hypertension,carcinoma, and other physiologic/pathologic problems.

[0135] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 2 1143 base pairs nucleic acid single linear cDNA Liver 86700 1ATGAGAAGTC ATACCATAAC AATGACGACA ACTTCAGTCA GCAGCTGGCC TTACTCCTCC 60CACAGAATGC GCTTTATAAC CAATCATAGC GACCAACCGC CACAAAACTT CTCAGCAACA 120CCAAATGTTA CTACCTGTCC CATGGATGAA AAATTGCTAT CTACTGTGTT AACCACATCC 180TACTCTGTTA TTTTCATCGT GGGACTGGTT GGGAACATAA TCGCCCCCTA TGTATTTCTG 240GGTATTCACC GTAAAAGAAA TTCCATTCAA ATTTATCTAC TTAACGTAGC CATTGCAGAC 300CTCCTACTCA TCTTCTGCCT CCCTTTCCGA ATAATGTATC ATATTAACCA AAACAAGTGG 360ACACTAGGTG TGATTCTGTG CAAGGTTGTG GGAACACTGC TTTATATGAA CATGTACATT 420AGCATTATTT TGCTTGGATT CATCAGTTTG GATCGCTATA TAAAAATTAA TCGGTCTATA 480CAGCAACGGA AGGCAATAAC AACCAAACAA AGTATTTATG TCTGTTGTAT AGTATGGATG 540CTTGCTCTTG GTGGATTCCT AACTATGATT ATTTTAACAC TTAAGAAAGG AGGGCATAAT 600TCCACAATGT GTTTCCATTA CAGAGATAAG CATAACGCAA AAGGAGAAGC CATTTTTAAC 660TTCATTCTTG TGGTAATGTT CTGGCTAATT TTCTTACTAA TAATCCTTTC ATATATTAAG 720ATTGGGAAGA ATCTATTGAG GATTTCTAAA AGGAGGTCAA AATTTCCTAA TTCTGGTAAA 780TATGCCACTA CAGCTCGTAA CTCCTTTATT GTACTTATCA TTTTTACTAT ATGTGTGGGT 840CCCTATCATG CCTTTCGATT CATCTACATT TCTTCACAGC TAAATGTATC ATCTTGCTAC 900TGGAAAGAAA TTGTTCACAA AACCAATGAG ATCATGCTGG TTCTCTCATC TTTCAATAGT 960TGGTTAGATC CAGTCATGTA TTTCCTGATG TCCAGTAACA TTCGCAAAAT AATGTGCCAA 1020CTTCTTTTTA GACGATTTCA AGGTGAACCA AGTAGGAGTG AAAGCACTTC AGAATTTAAA 1080CCAGGATACT CCCTGCATGA TACATCTGTG GCAGGGAAAA TACAGTCTAG TTCTGAAAGT 1140ACT 1143 381 amino acids amino acid linear protein 2 Met Arg Ser His ThrIle Thr Met Thr Thr Thr Ser Val Ser Ser Trp 1 5 10 15 Pro Tyr Ser SerHis Arg Met Arg Phe Ile Thr Asn His Ser Asp Gln 20 25 30 Pro Pro Gln AsnPhe Ser Ala Thr Pro Asn Val Thr Thr Cys Pro Met 35 40 45 Asp Glu Lys LeuLeu Ser Thr Val Leu Thr Thr Ser Tyr Ser Val Ile 50 55 60 Phe Ile Val GlyLeu Val Gly Asn Ile Ile Ala Pro Tyr Val Phe Leu 65 70 75 80 Gly Ile HisArg Lys Arg Asn Ser Ile Gln Ile Tyr Leu Leu Asn Val 85 90 95 Ala Ile AlaAsp Leu Leu Leu Ile Phe Cys Leu Pro Phe Arg Ile Met 100 105 110 Tyr HisIle Asn Gln Asn Lys Trp Thr Leu Gly Val Ile Leu Cys Lys 115 120 125 ValVal Gly Thr Leu Leu Tyr Met Asn Met Tyr Ile Ser Ile Ile Leu 130 135 140Leu Gly Phe Ile Ser Leu Asp Arg Tyr Ile Lys Ile Asn Arg Ser Ile 145 150155 160 Gln Gln Arg Lys Ala Ile Thr Thr Lys Gln Ser Ile Tyr Val Cys Cys165 170 175 Ile Val Trp Met Leu Ala Leu Gly Gly Phe Leu Thr Met Ile IleLeu 180 185 190 Thr Leu Lys Lys Gly Gly His Asn Ser Thr Met Cys Phe HisTyr Arg 195 200 205 Asp Lys His Asn Ala Lys Gly Glu Ala Ile Phe Asn PheIle Leu Val 210 215 220 Val Met Phe Trp Leu Ile Phe Leu Leu Ile Ile LeuSer Tyr Ile Lys 225 230 235 240 Ile Gly Lys Asn Leu Leu Arg Ile Ser LysArg Arg Ser Lys Phe Pro 245 250 255 Asn Ser Gly Lys Tyr Ala Thr Thr AlaArg Asn Ser Phe Ile Val Leu 260 265 270 Ile Ile Phe Thr Ile Cys Val GlyPro Tyr His Ala Phe Arg Phe Ile 275 280 285 Tyr Ile Ser Ser Gln Leu AsnVal Ser Ser Cys Tyr Trp Lys Glu Ile 290 295 300 Val His Lys Thr Asn GluIle Met Leu Val Leu Ser Ser Phe Asn Ser 305 310 315 320 Trp Leu Asp ProVal Met Tyr Phe Leu Met Ser Ser Asn Ile Arg Lys 325 330 335 Ile Met CysGln Leu Leu Phe Arg Arg Phe Gln Gly Glu Pro Ser Arg 340 345 350 Ser GluSer Thr Ser Glu Phe Lys Pro Gly Tyr Ser Leu His Asp Thr 355 360 365 SerVal Ala Gly Lys Ile Gln Ser Ser Ser Glu Ser Thr 370 375 380

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequence SEQID NO:2, b) a polypeptide comprising a naturally occurring human variantof the amino acid sequence of SEQ ID NO:2, c) a biologically activefragment of a polypeptide having the amino acid sequence of SEQ ID NO:2,and d) an immunogenic fragment of a polypeptide having the amino acidsequence of SEQ ID NO:2.
 2. An isolated polypeptide of claim 1comprising the amino acid sequence of SEQ ID NO:2.
 3. An isolatedpolynucleotide encoding a polypeptide selected from the group consistingof: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2,b) a polypeptide comprising a naturally occurring human variant of theamino acid sequence of SEQ ID NO:2, c) a biologically active fragment ofa polypeptide having the amino acid sequence of SEQ ID NO:2, and d) animmunogenic fragment of a polypeptide having the amino acid sequence ofSEQ ID NO:2.
 4. An isolated polynucleotide of claim 3 encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:2.
 5. Anisolated polynucleotide of claim 4 comprising the polynucleotidesequence of SEQ ID NO:1.
 6. A recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide of claim
 3. 7. Acell transformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method of producing a polypeptide encoded by a polynucleotide ofclaim 3, the method comprising: a) culturing a cell under conditionssuitable for expression of the polypeptide, wherein said cell istransformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide of claim 3, and b) recovering the polypeptide soexpressed.
 10. A method of claim 9, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO:2.
 11. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 12. An isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1,b) a polynucleotide comprising a naturally occurring human variant ofthe polynucleotide sequence of SEQ ID NO:1, c) a polynucleotidecomplementary to a polynucleotide of a), d) a polynucleotidecomplementary to a polynucleotide of b), and e) an RNA equivalent ofa)-d).
 13. An isolated polynucleotide comprising at least 60 contiguousnucleotides of a polynucleotide of claim
 12. 14. A method of detecting atarget polynucleotide in a sample, said target polynucleotide having asequence of a polynucleotide of claim 12, the method comprising: a)hybridizing the sample with a probe comprising at least 20 contiguousnucleotides comprising a sequence complementary to said targetpolynucleotide in the sample, and which probe specifically hybridizes tosaid target polynucleotide, under conditions whereby a hybridizationcomplex is formed between said probe and said target polynucleotide orfragments thereof, and b) detecting the presence or absence of saidhybridization complex, and, optionally, if present, the amount thereof.15. A method of claim 14, wherein the probe comprises at least 60contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises the aminoacid sequence of SEQ ID NO:2.
 19. A method for treating a disease orcondition associated with decreased expression of functional HUMTHRR,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HUMTHRR, comprising administering to a patient in need ofsuch treatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HUMTHRR, comprising administering to apatient in need of such treatment a composition of claim
 24. 26. Amethod of screening for a compound that specifically binds to thepolypeptide of claim 1, the method comprising: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 27. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, themethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an un treated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of HUMTHRR in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HUMTHRR in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofHUMTHRR in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of the amino acid sequence of SEQ ID NO:2, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibodies from said animal, and c) screening theisolated antibodies with the polypeptide, thereby identifying apolyclonal antibody which binds specifically to a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2.
 37. A polyclonal antibodyproduced by a method of claim
 36. 38. A composition comprising thepolyclonal antibody of claim 37 and a suitable carrier.
 39. A method ofmaking a monoclonal antibody with the specificity of the antibody ofclaim 11, the method comprising: a) immunizing an animal with apolypeptide consisting of the amino acid sequence of SEQ ID NO:2, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibody producing cells from the animal, c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide comprising the amino acidsequence of SEQ ID NO:2.
 40. A monoclonal antibody produced by a methodof claim
 39. 41. A composition comprising the monoclonal antibody ofclaim 40 and a suitable carrier.
 42. The antibody of claim 11, whereinthe antibody is produced by screening a Fab expression library.
 43. Theantibody of claim 11, wherein the antibody is produced by screening arecombinant immunoglobulin library.
 44. A method of detecting apolypeptide comprising the amino acid sequence of SEQ ID NO:2 in asample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) detecting specific binding, wherein specificbinding indicates the presence of a polypeptide comprising the aminoacid sequence of SEQ ID NO:2 in the sample.
 45. A method of purifying apolypeptide comprising the amino acid sequence of SEQ ID NO:2 from asample, the method comprising: a) incubating the antibody of claim 11with a sample under conditions to allow specific binding of the antibodyand the polypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising the amino acid sequence ofSEQ ID NO:2.
 46. A microarray wherein at least one element of themicroarray is a polynucleotide of claim
 13. 47. A method of generatingan expression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:2.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO: 1.